Modelling of the high pressure–temperature eﬀects on naringin hydrolysis based on response surface methodology Joa ˜o Marques a , Helder J. Vila-Real a , Anto ´nio J. Alfaia a,b , Maria H.L. Ribeiro a,b, * a Faculdade de Farma ´ cia, Universidade de Lisboa, Av. Gama Pinto, 1649-003 Lisboa, Portugal b Centro de Estudos de Cie ˆncias Farmace ˆuticas, Faculdade de Farma ´ cia, Universidade de Lisboa, Av. Gama Pinto, 1649-003 Lisboa, Portugal Received 3 December 2006; received in revised form 12 February 2007; accepted 3 April 2007 Abstract The aim of this study was the modelling, under high pressure, of naringin hydrolysis by naringinase. Response surface methodology (RSM) was used to compare the eﬀects of the selected variables on the bioconversion under study. The combined action of temperature (13–61 °C) and pressure (80–216 MPa) on the catalytic activity of naringinase was investigated at pH 4.0 using naringin as the substrate. The choice of experimental domains resulted from preliminary studies. Naringinase activity, for naringin hydrolysis at pH 4.0, could be described by a convex surface with a maximum of 0.13 mM min 1 , at 41 °C and 158 MPa. After 1 h of reaction time, reducing sugars production could also be described by a convex surface, with a maximum reducing sugars concentration of 8 mM at 38 °C and 168 MPa. The interaction temperature–pressure had a signiﬁcant eﬀect on both naringinase activity and reducing sugar formation after 1 h. Under the optimized conditions, the naringin hydrolysis by naringinase was evaluated. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: High pressure–temperature eﬀects; Naringin; Naringinase; RSM 1. Introduction In recent years, biocatalysis oﬀers a number of key advantages over chemical synthesis when working on com- plex molecules, advantages based on the chemo-, regio- and stereoselectivity of enzymes and conditions. The use of high pressure for the enzymatic synthesis of pharmacological molecules, namely ﬂavonoids, is interesting. Flavonoids are a class of plant speciﬁc natural products involved in nodulation, UV protection and host defence in plants and various health-promoting eﬀects in humans, such as antitu- mor, prevention of cardiovascular diseases, inhibition of enzymatic lipoperoxidation, and anti-inﬂammatory and antithrombotic eﬀects (Di Carlo, Mascolo, Izzo, & Cap- asso, 1999). Flavonoids are potential chemopreventive agents, due to their activities as inhibitors of enzymes involved in the biotransformation of precarcinogens. Naringinase, an a-rhamnopyranosidase, hydrolyze naringin to naringenin which has important biological eﬀects, such as anti-oxidant, anti-ulcer, anti-mutagenic, anti-inﬂamma- tory, anti-thrombotic, vasodilator, and anticancer eﬀects, inhibiting the proliferation of breast cancer and delaying mammary tumorigenesis (Nishino, Ngao, Fujili, & Sugim- ura, 1983). High pressure can be used to modulate both the stability and activity of several enzymes (Heremans & Smeller, 1998), can, also modify the catalytic behaviour of enzymes by changing the rate-limiting step or modulating the selec- tivity of the enzyme (Mozhaev, Heremans, Frank, Mans- son, & Balny, 1996). It was found that pressure could 0308-8146/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2007.04.012 Abbreviations: CCRD, central composite rotatable design; CV, coeﬃcient of variation; RSM, response surface methodology; R 2 , determination co- eﬃcient (quadratic correlation coeﬃcient); R 2 adj , adjusted determination coeﬃcient; P, pressure; T, temperature. * Corresponding author. Address: Faculdade de Farma ´cia, Universid- ade de Lisboa, Av. Gama Pinto, 1649-003 Lisboa, Portugal. Tel.: +351 21 7946453; fax: +351 21 7946470. E-mail address: mhribeiro@ﬀ.ul.pt (M.H.L. Ribeiro). www.elsevier.com/locate/foodchem Food Chemistry 105 (2007) 504–510 Food Chemistry